Method for determining quantity of biological material in tissue section

ABSTRACT

An object of the present invention is to provide a method of detecting a specific tissue or cell in a sample tissue section and accurately specifying both the position(s) and amount of a biological substance of interest that is expressed on the specific tissue or cell. The method of quantifying a biological substance in a tissue section according to the present invention comprises: (1) performing bright-field observable immunostaining that specifically stains a first biological substance in the tissue section (first immunostaining); (2) performing immunostaining with a fluorescent substance-containing nanoparticle that specifically stains a second biological substance in the tissue section (second immunostaining); (3) specifying the expression position(s) of the second biological substance in the tissue section by comparing the position of a stained image of the first immunostaining and the position of a stained image of the second immunostaining; and (4) determining the expression amount of the second biological substance by measuring the fluorescence intensity of the stained image of the second immunostaining.

TECHNICAL FIELD

The present invention relates to a method of quantifying a biologicalsubstance in a tissue section. More particularly, the present inventionrelates to a method of quantifying a biological substance in a tissuesection using immunostaining.

BACKGROUND ART

In the field of pathological diagnosis, it is useful to detect aspecific cell(s) or tissue from a specimen of a sampled tissue sectionand quantify the expression level of a substance associated with alesion on the specific cell(s) or tissue, and methods utilizingimmunohistochemistry have been examined for this purpose.

Immunohistochemistry (IHC) is widely known as a histological(histochemical) technique for detecting an antigen in a tissue sampleusing an antibody. This immunohistochemistry is sometimes referred to as“immunostaining” or the like (hereinafter, the term “immunohistochemicalstaining” may also be used for immunohistochemistry) because it involvesa color-developing operation in order to visualize an antigen-antibodyreaction that is intrinsically invisible. Due to this characteristicfeature of visualizing the location of an antigen-antibody reaction,immunohistochemistry is widely used in the fields of medicine and lifechemistry for the purpose of detecting the location of a biologicalsubstance in a tissue sample.

In IHC, as a method of visualizing the location of an antigen-antibodyreaction, a staining method that can be observed in a bright field iswidely used and, specifically, a technique using a substrate that isconverted into a pigmentary substance by an enzyme is commonly used. Forexample, in clinical scenes, it is widely performed to stain and thusvisualize an antibody bound to an antigen of interest in a tissue sampleusing peroxidase (POD) and diaminobenzidine (DAB) and to detect theexpression amount of the specific antigen by bright-field observationthrough the thus visualized antibody. This bright-field observation isadvantageous in that, as compared to the below-described method using alight-emitting substance, more detailed information on a target moleculecan be obtained by comprehensively judging the information obtained inan analog manner such as staining color. As a method of distinguishingcytoma and specifying the position of the cytoma in a specimen usingsuch immunostaining, an immunostaining technology which uses a markerspecifically expressed in cells as a target has been generally employed.

As a method of evaluating the expression level of a biological substanceof interest in a tissue section with high accuracy usingimmunohistochemistry, there is known a method which comprises performingimmunostaining with a fluorescent substance-containing nanoparticle,detecting the thus generated fluorescent bright dots and then evaluatingthe expression level of a biological substance (Patent Document 1). Inthis method, the brightness distribution corresponding to the biologicalsubstance of interest can be measured by the use of the fluorescentsubstance-containing nanoparticle; however, it is difficult to specifythe positional relationship between a specific tissue or cell in atissue section and the biological substance of interest and thus tomeasure the brightness distribution by selecting the biologicalsubstance of interest expressed on the specific tissue or cell.

In addition, a method of simultaneously performing bright-fieldobservable staining by an enzyme antibody method (DAB staining that usesan enzyme reaction) and fluorescent dye staining by a fluorescentantibody method (staining that uses a fluorescent dye) on a singletissue section has been reported (Non-patent Documents 1 and 2). In thismethod, although a biological substance of interest is quantified usinga fluorescent dye, since a sufficient fluorescence intensity cannot beobtained at those spots where the biological substance is expressed in asmall amount, it is difficult to quantify the biological substance ofinterest in some cases. For instance, in an example in whichKi-67-positive cells were stained by an enzyme antibody method andcytokeratin was fluorescently stained by a fluorescent antibody method,it is reported that there were cases where fluorescence observation ofcytokeratin was difficult due to inadequate amount of fluorescence(Non-patent Document 2).

As a method of quantifying a protein by multiple fluorescent staining,there has also been proposed a method that comprises staining a proteinof interest to be quantified and a reference protein with differentfluorescent dye-labeled antibodies, measuring the total fluorescenceintensities of the respective proteins and then quantifying the proteinof interest based on the ratio of the thus measured total fluorescenceintensities (Patent Document 2).

As described above, with regard to a method of detecting a specifictissue or cell in a sampled tissue section and quantifying a biologicalsubstance of interest that is expressed on the specific tissue or cell,there is a demand for a method of more accurately specifying both theexpression position (s) and the expression amount.

PRIOR ART REFERENCES Patent Documents

[Patent Document 1] JP-A-2013-088296

[Patent Document 2] JP-A-2008-268167

Non-Patent Documents

[Non-patent Document 1] Tuominen, V.; Ruotoisteumaki, S.; Viitanen, A.;Jumppanen, M. and Isola, J; Breast Cancer Research 2010, 12:R56,“ImmunoRatio-F: a publicly available web application for quantitativeimage analysis of estrogen receptor (ER), progesterone receptor (PR),and Ki-67”

[Non-patent Document 2] “URL:http://mitel.dimi.uniud.it/tp2012/presentations/A8-Isola.pdf” VilppuTuominen, Sofia Heinonen, Jorma Isola, “ImmunoRatio-F: image analysis ofKi-67 using cytokeratin immunofluorescence correction”

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method of detecting aspecific tissue or cell in a sampled tissue section and accuratelyspecifying both the position(s) and amount of a biological substance ofinterest that is expressed on the specific tissue or cell.

Technical Solution

The present inventors discovered that, by staining a biologicalsubstance for specifying a specific tissue or cell (first biologicalsubstance) and a biological substance of interest for which theexpression amount is quantified (second biological substance) bydifferent methods in the same tissue section and comparing the positionsof the stained images of these biological substances, the expressionpositions of the biological substance of interest (second biologicalsubstance) can be accurately specified; and that, by staining thebiological substance of interest (second biological substance) with afluorescent substance-containing nanoparticle, the biological substanceof interest (second biological substance) can be accurately quantifiedeven when the expression amount thereof is small, thereby completing thepresent invention.

That is, the present invention comprises the following items.

[1] A method of quantifying a biological substance in a tissue section,the method comprising:

(1) performing bright-field observable immunostaining that specificallystains a first biological substance in the tissue section (firstimmunostaining);

(2) performing immunostaining with a fluorescent substance-containingnanoparticle that specifically stains a second biological substance inthe tissue section (second immunostaining);

(3) specifying the expression position(s) of the second biologicalsubstance in the tissue section by comparing the position of a stainedimage of the first immunostaining and position of a stained image of thesecond immunostaining; and

(4) determining the expression amount of the second biological substanceby measuring the fluorescence intensity of the stained image of thesecond immunostaining.

[2] The method according to [1], wherein the fluorescence intensity ofthe stained image of the second immunostaining is measured at aposition(s) where the stained image of the first immunostaining and thatof the second immunostaining overlap with each other.

[3] The method according to [1] or [2], wherein the first immunostainingand the second immunostaining are performed on the same tissue section.

Advantageous Effects of Invention

According to the present invention, a method of detecting a specifictissue or cell in a tissue section and accurately specifying both theposition(s) and amount of a biological substance of interest that isexpressed on the specific tissue or cell can be provided.

That is, by detecting a specific tissue or cell based on a stained imageof a first biological substance that can be observed in a bright fieldand measuring the bright spots and the brightness distribution in afluorescently stained image of a second biological substance for thoseparts where the position of the stained image of the first biologicalsubstance and the position of the fluorescently stained image (thenumber of bright spots and brightness distribution) of the secondbiological substance overlap with each other, the second biologicalsubstance (biological substance of interest) expressed on the specifictissue or cell can be selectively quantified. Further, since the secondbiological substance is stained with a fluorescent substance-containingnanoparticle, a high detection performance (sensitivity) can be attainedeven when the expression amount of the second biological substance issmall.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a stained image obtained by the bright-field observableimmunostaining performed in Example 2 of the present invention (tissuesection: human liver tissue, immunostaining: CD31 antibody, dye: DAB).

FIG. 2 shows a stained image obtained by the immunostaining with afluorescent substance-containing nanoparticle that was performed inExample 2 of the present invention (tissue section: the same human livertissue as shown in FIG. 1, immunostaining: VEGFR2 antibody, dye:SulfoRhodamine 101-containing nanoparticle).

MODE FOR CARRYING OUT THE INVENTION

The mode for carrying out of the present invention will now be describedin detail; however, the present invention is not restricted thereto.

1. Substance to be Detected and Substance to be Quantified

The present invention is a method of detecting a specific tissue or cell(hereinafter, referred to as “detection subject”) in a sampled tissuesection and accurately specifying both the position(s) and amount of abiological substance of interest (hereinafter, referred to as “substanceto be quantified”) that is expressed on the detection subject. In thepresent invention, a combination of the subject tissue section,detection subject and substance to be quantified can be selected inaccordance with the examination purpose. For example, quantification ofVEGFR-1, VEGFR-2, VEGFR-3 or the like expressed on vascular endothelialcells in a liver tissue section, quantification of VEGFR-1, VEGFR-2,VEGFR-3 or the like expressed on lymphatic endothelial cells in anesophageal tissue section, or quantification of Ki67 expressed onepithelial cells in a breast tissue section can be performed.

2. Tissue Section

The tissue section in the present invention is not particularlyrestricted as long as it is a section to which an immunostaining methodcan be applied, and such a tissue section can be prepared by a knownmethod. For example, a paraffin-embedded section or the like that iswidely used as a pathological section can be used as the tissue section.

3. Immunostaining

In the present invention, bright-field observable immunostaining thatspecifically stains a first biological substance in a tissue section(first immunostaining) and immunostaining with a fluorescentsubstance-containing nanoparticle that specifically stains a secondbiological substance in the tissue section (second immunostaining) areperformed. In other words, the detection subject is detected based on astained image obtained by the first immunostaining and the substance tobe quantified is quantified based on the bright spots and the brightnessdistribution of a fluorescently stained image obtained by the secondimmunostaining.

In this case, these immunostainings are each performed using a “labeledprobe” in which an antibody that specifically binds to the first orsecond biological substance (hereinafter, referred to as “targetsubstance”) and a visualizable substance (hereinafter, referred to as“label”) are bound with each other. In other words, in the labeledprobe, an antibody to a target substance is bound with a label. Here,the method of binding the antibody and the label is not particularlyrestricted and, in addition to a case where they are directly bound witheach other, the present invention also encompasses those cases where theantibody and the label are bound through a secondary antibody.

Hereinafter, a labeled probe for performing the first immunostaining isreferred to as “the first labeled probe”, and a labeled probe forperforming the second immunostaining is referred to as “the secondlabeled probe”.

In the present invention, it is required that the first labeled probeand the second labeled probe do not inhibit each other'santigen-antibody reaction with their respective target substance.

(1) First Labeled Probe (a) First Biological Substance

The first biological substance is a target substance of immunostaining(the first immunostaining) contained in the detection subject. As thefirst biological substance, a substance which functions as an antigen onthe tissue or cell that is the detection subject may be selected so thatbright-field observable staining is performed on the detection subjectto such an extent that conforms to the examination purpose. For example,the first biological substance may be CD31, CD34 or the like forimmunostaining of vascular endothelial cells, podoplanin or the like forimmunostaining of lymphatic endothelial cells, or cytokeratin or thelike for immunostaining of epithelial cells.

(b) Antibody Binding to First Biological Substance

An antibody that specifically binds to the first biological substancecan be obtained by an ordinary method.

(c) Label

A label is used for visualizing a labeled probe bound to a targetsubstance on a tissue section, and the label contained in the firstlabeled probe is a label for performing bright-field observablestaining.

In the present invention, the term “bright-field observable staining”refers to staining that is directly visualized in a form visible underan ordinary light microscope (in other words, staining that reflectsvisible light) without excitation by an energy applied from outside. Thephrase “directly visualized in a form visible” refers to achieving astate where the site(s) of specific binding reaction between a targetsubstance and a probe can be directly observed without performing anysecondary operation such as development.

Examples of such a label contained in the first labeled probe are asfollows.

(Pigmentation-Inducing Label)

Examples of a label that enables bright-field observation of the presentinvention include substances that induce pigmentation(pigmentation-inducing labels), in other words, enzymes that modify asubstrate to cause the generation of a pigmentary chemical species.Examples of such enzymes include peroxidases such as horseradishperoxidase (HRP) and other enzymes such as alkaline phosphatase (ALP)and glucosidase.

(Substrate Used for Pigmentation)

As a substrate to be converted into a pigmentary substance by an enzymeas described above, a substrate that is commonly used as a chromogenicsubstrate in conventionally known assays based on a chromogenicsubstrate conversion method can be used. Examples of such a substrateinclude, but not limited to, substrates for oxidoreductases such as asubstrate for horseradish peroxidase (HRP) , substrates for phosphatasessuch as a substrate for alkaline phosphatase (ALP), and substrates forglycosidases such as a substrate for β-galactosidase.

Specific examples of a substrate used for an enzyme reaction by HRPinclude 3,3′-diaminobenzidine (DAB), 3-p-hydroxyphenylpropionic acid(HPPA), ECL plus (trademark) and4-chloro-1-naphthol/4-chloronaphthalen-1-ol. Thereamong, DAB ispreferably used because it is widely used from the standpoints of itscolor difference from hematoxylin (blue) , which is commonly used fornuclear staining, and storage stability.

Examples of a substrate used for an enzyme reaction by alkalinephosphatase (ALP) include a 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium salt (BCIP/NBT), 4-methylumbelliferylphosphate (MUP), 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP),AttoPhos (registered trademark), and9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAOP).

Examples of a substrate used for an enzyme reaction by β-galactosidaseinclude 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal),9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)-β-D-galactop yranoside(DDAOG), and 4-methylumbelliferyl-β-D-galactoside (MUG).

(d) First Labeled Probe

The first labeled probe can be obtained by binding the above-describedantibody that binds to the first biological substance with theabove-described label. In this case, the method of binding the antibodyand the label is not particularly restricted and, as described above, inaddition to a case where they are directly bound with each other, thepresent invention also encompasses those cases where the antibody andthe label are bound through a secondary antibody.

Such binding of the antibody and the label can be achieved by bindingthe label to the antibody in accordance with a commonly used method.Examples of a specific labeling method include a method of labeling theantibody through an antibody (secondary antibody) having a specificaffinity for the antibody; a biotin-avidin method; a method thatutilizes the coupling reaction between a thiol group and a maleimidegroup; a method that uses an existing chemical linker; a cross-linkingreaction method that uses a cross-linking agent (such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)); and an ionic bondmethod (see the below-described Examples).

(2) Second Labeled Probe (a) Second Biological Substance

The second biological substance is a target substance of immunostaining(the second immunostaining) contained in the substance to be quantified.A substance which functions as an antigen on the substance to bequantified can be selected so that quantification that conforms to theexamination purpose is performed. However, the substance to bequantified is generally a protein and, in that case, the substance to bequantified itself is the second biological substance. Examples of thesecond biological substance include vascular endothelial growth factorreceptors (VEGFR-1, VEGFR-2 and VEGFR-3) and cell growth-relatedproteins such as Ki-67.

(b) Antibody Binding to Second Biological Substance

An antibody that specifically binds to the second biological substancecan be obtained by an ordinary method.

(c) Label

A label is used for visualizing a labeled probe bound to a targetsubstance on a tissue section, and the label contained in the secondlabeled probe is a fluorescent substance-containing nanoparticle.

The term “fluorescent substance-containing nanoparticle” refers to anano-sized particle having a structure in which two or more (two or moremolecules) of a fluorescent substances are contained in a particle(matrix) made of an organic or inorganic material. In the presentinvention, the fluorescent substance is a fluorescent dye or afluorescent nanoparticle, and examples thereof include fluorescentdye-containing nanoparticles and fluorescent nanoparticle-containingnanoparticles. The fluorescent substance-containing nanoparticle(fluorescent dye-containing nanoparticle or fluorescentnanoparticle-containing nanoparticle) used in the present invention canbe prepared by a known method by selecting, in accordance with theintended purpose thereof, a fluorescent dye or fluorescent nanoparticleused as a fluorescent substance and a particle-forming organic orinorganic material as raw materials.

Examples of the particle-forming organic or inorganic material includethose which are capable of stably containing a fluorescent substance,such as polystyrene, polyamide, polylactic acid, polyacrylonitrile,polyglycidyl methacrylate, polymelamine, polyurea, polybenzoguanamine,polyfuran, polyxylene, phenol resins, polysaccharides and silica. When afluorescent substance is incorporated into such a particle,deterioration thereof caused by irradiation with an excitation light isless likely to occur (superior light resistance is attained) as comparedto a case where a fluorescent dye is used by itself and, byincorporating plural fluorescent substances in one particle, thefluorescence intensity (brightness) of the light emitted from oneparticle can be increased.

(Fluorescent Dye-Containing Nanoparticle)

The fluorescent dye-containing nanoparticle contains two or morefluorescent dye molecules in one particle described above. Thefluorescent dye to be contained is not particularly restricted and canbe selected in accordance with the intended excitation light,fluorescence wavelength and the like.

The fluorescent dye to be contained can be selected from, for example,rhodamine-based dye molecules, squarylium-based dye molecules,cyanine-based dye molecules, aromatic ring-based dye molecules,oxazine-based dye molecules, carbopyronine-based dye molecules andpyrromethene-based dye molecules. Alternatively, the fluorescent dye tobe contained can also be selected from, for example, Alexa Fluor(registered trademark, manufactured by Invitrogen)-based dye molecules,BODIPY (registered trademark, manufactured by Invitrogen)-based dyemolecules, Cy (registered trademark, manufactured by GEHealthcare)-based dye molecules, DY (registered trademark, DyomicsGmbH)-based dye molecules, HiLyte (registered trademark, manufactured byAnaSpec Inc.)-based dye molecules, DyLight (registered trademark,manufactured by Thermo Fisher Scientific K.K.)-based dye molecules, ATTO(registered trademark, manufactured by ATTO-TEC GmbH)-based dyemolecules and MFP (registered trademark, manufactured by Mobitec Co.,Ltd.)-based dye molecules. The generic names of these dye molecules aredesignated based on the main structure (skeleton) or registeredtrademark of the respective compounds; therefore, those of ordinaryskill in the art can properly understand the scope of the fluorescentdyes belonging to the respective generic names without having to bearundue trial and error.

Specific examples of the rhodamine-based dye molecules include5-carboxy-rhodamine, Texas Red and SulfoRhodamine 101. Specific examplesof the squarylium-based dye molecules include SRfluor 680-carboxylate.Specific examples of the cyanine-based dye molecules include1-butyl-2-[5-(1-butyl-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene)-penta-1,3-dienyl]-3,3-dimethyl-3H-indoliumhexafluorophosphate. Specific examples of the aromatic ring-based dyemolecules includeN,N-bis-(2,6-diisopropylphenyl)-1,6,7,12-(4-tert-butylphenoxy)-perylen-3,4,9,10-tetracarbonaciddiimide. Specific examples of the oxazine-based dye molecules includeCresyl violet. Specific examples of the carbopyronine-based dyemolecules include CARBOPYRONIN 149. Specific examples of thepyrromethene-based dye molecules include PYRROMETHENE 650.

Further, specific examples of the Alexa Fluor-based dye moleculesinclude Alexa Fluor 555 (manufactured by Invitrogen). Specific examplesof the BODIPY-based dye molecules include BODIPY FL (manufactured byInvitrogen). Specific examples of the Cy-based dye molecules include Cy3.5 (manufactured by GE Healthcare). Specific examples of the DY-baseddye molecules include DY-590 (manufactured by Dyomics GmbH). Specificexamples of the HiLyte-based dye molecules include HiLyte 594(manufactured by AnaSpec Inc.). Specific examples of the DyLight-baseddye molecules include DyLight 594 (manufactured by Thermo FisherScientific K.K.). Specific examples of the ATTO-based dye moleculesinclude ATTO 590 (manufactured by ATTO-TEC GmbH). Specific examples ofthe MFP-based dye molecules include MFP 590 (manufactured by MobitecCo., Ltd.).

Examples of other dyes include C-phycocyanin, phycocyanin, APC(allophycocyanin), APC-XL and NorthernLights 637 (all of which aremanufactured by R&D Systems, Inc.).

Further, specific examples of the fluorescent dye also includederivatives of the above-described dyes (which can function as afluorescent dye, such as known derivatives).

The method of producing the fluorescent dye-containing nanoparticle isnot particularly restricted, and the fluorescent dye-containingnanoparticle is produced by a commonly used method. Examples thereofinclude a method in which, after binding a fluorescent dye molecule (s)to a resin monomer used as a particle raw material, a particle issynthesized by polymerizing the resin monomer; and a method ofintroducing a fluorescent dye(s) to a polymerized resin particle throughadsorption or binding; a method in which a resin monomer and afluorescent dye(s) are mixed and then polymerization of the resinmonomer and binding of the fluorescent dye(s) are performedsimultaneously. In the fluorescent dye-containing nanoparticles producedby these methods, plural fluorescent dye molecules are usually containedin one particle.

The average particle size of the fluorescent dye-containing nanoparticleis not particularly restricted; however, it is usually 10 to 500 nm,preferably 50 to 200 nm. Further, the variation coefficient whichindicates the variation in the particle size is also not particularlyrestricted; however, it is usually 20% or less, preferably 5 to 15%.

It is noted here that the particle size of a fluorescent dye-containingnanoparticle can be determined by taking an electron micrograph thereofusing a scanning electron microscope (SEM), measuring thecross-sectional area of the fluorescent dye-containing nanoparticle andthen determining the particle size as the diameter of a circular areacorresponding to the measured value (area-equivalent circle diameter).With regard to the average particle size (average particle diameter) andthe variation coefficient of a group of fluorescent dye-containingnanoparticles, after measuring the particle size (particle diameter) fora sufficient number (for example, 1,000) of the fluorescentdye-containing nanoparticles in the above-described manner, the averageparticle size is calculated as the arithmetic mean of the measuredvalues and the variation coefficient is calculated by the followingequation: 100×(standard deviation of particle size)/(average particlesize).

(Fluorescent Nanoparticle-Containing Particle)

The fluorescent nanoparticle-containing particle contains two or morefluorescent nanoparticles in one particle described above. Thefluorescent nanoparticles to be contained are not particularlyrestricted and can be selected in accordance with the intendedexcitation light, fluorescence wavelength and the like.

The fluorescent nanoparticles to be contained have a particle size of 1to 500 nm, preferably 10 to 200 nm. These fluorescent nanoparticles arecomposed of a semiconductor or fluorophore. As the semiconductor, forexample, a group II-VI semiconductor such as ZnSe, ZnTe, CdSe, CdTe,PbS, PbSe or PbTe, or a group III-V semiconductor such as AlAs, AlSb,GaP, GaAs, GaSb, InP, InAs or InSb can be used. From the standpoint oftoxicity, GaP or InP can be suitably used. In the fluorophore, forexample, Y₂O₃, YVO₄, ZnO or ZnS can be used as the matrix and Eu or Ndcan be used as the emission center. An excitation wavelength suitablefor observation is set by adjusting the particle size, matrixcomposition and impurity amount of the fluorescent nanoparticles.

The method of producing the fluorescent nanoparticle-containingnanoparticle by incorporating such fluorescent nanoparticles into theabove-described particle is not particularly restricted, and thefluorescent nanoparticle-containing nanoparticle is produced by acommonly used method. Examples thereof include a method in which, afterbinding fluorescent nanoparticles to a resin monomer used as a particleraw material, a particle is synthesized by polymerizing the resinmonomer; a method of introducing fluorescent nanoparticles to apolymerized resin particle through adsorption or binding; and a methodin which a resin monomer and fluorescent nanoparticles are mixed andthen polymerization of the resin monomer and binding of the fluorescentnanoparticles are performed simultaneously. In the fluorescentnanoparticle-containing nanoparticles produced by these methods, pluralfluorescent nanoparticles are usually contained in one particle. Anexcitation wavelength suitable for observation is set by adjusting theparticle size, matrix composition and impurity amount of the containedfluorescent nanoparticles. The fluorescent nanoparticle-containingparticle has a particle size of 10 to 500 nm, preferably 50 to 200 nm.

The particle size of a fluorescent nanoparticle-containing nanoparticlecan be determined in the same manner as in the case of theabove-described fluorescent dye-containing nanoparticle.

(d) Second Labeled Probe

The second labeled probe can be obtained by binding the above-describedantibody that binds to the second biological substance with theabove-described label. In this case, the method of binding the antibodyand the label is not particularly restricted and, as described above, inaddition to a case where they are directly bound with each other, thepresent invention also encompasses those cases where the antibody andthe label are bound through a secondary antibody.

Such binding of the antibody and the label can be achieved by bindingthe label to the antibody in accordance with a commonly used method.Examples of a specific labeling method include a method of labeling theantibody through an antibody (secondary antibody) having a specificaffinity for the antibody; a biotin-avidin method; a method thatutilizes the coupling reaction between a thiol group and a maleimidegroup; a method that uses an existing chemical linker; a cross-linkingreaction method that uses a cross-linking agent (such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)); and an ionic bondmethod (see the below-described Examples).

(3) Immunostaining

In the present invention, the above-described first immunostaining andsecond immunostaining are performed on a tissue section. In this case,it is preferred that both of these immunostainings be performed on thesame tissue section so as to specify the position(s) at which thesubstance to be quantified is expressed on the detection subject bycomparing the positional relationship between the stained imagesobtained by the respective immunostainings; however, it is also possibleto perform each immunostaining on adjacent tissue sections that areobtained in the process of cutting out sections.

These immunostainings can each be performed by a commonly used method.

4. Comparisons of Positions of Stained Images

In the present invention, the positions of the second biologicalsubstance expressed on a tissue section are specified by comparing theposition of a stained image of the first immunostaining and the positionof a stained image of the second immunostaining. It is noted here that,as described above, the stained image of the first immunostaining can beobserved in a bright field and the stained image of the secondimmunostaining is a fluorescently stained image. Accordingly, thestained image of the first immunostaining is observed under a lightmicroscope, while the stained image of the second immunostaining isobserved under a fluorescence microscope. For the positional comparisonbetween these stained images, any method can be employed with noparticular restriction as long as the method is capable of determiningto which positions on the stained image of the first immunostaining thebright spots and brightness distribution obtained by the secondimmunostaining correspond. For example, by loading the stained image ofthe first immunostaining and the stained image of the secondimmunostaining to a computer and comparing the positional relationshipsbetween these images, the positional relationships of the bright spotsand brightness distribution obtained by the second immunostaining withrespect to the stained image of the first immunostaining can be judged.Examples of an analysis software for such process include a commerciallyavailable image analysis software, ImageJ.

Since the detection subject on a tissue section is specified by thefirst immunostaining, as long as the positional relationships betweenthe above-described stained images can be determined by positionalcomparison, the bright spots and brightness distribution obtained by theimmunostaining of the substance to be quantified (second biologicalsubstance) that is expressed on the detection subject in the tissuesection can be specified. In other words, the bright spots andbrightness distribution of the second immunostaining that are found atthe positions at which the stained image of the first immunostaining andthat of the second immunostaining overlap with each other are the brightspots and brightness distribution of the substance to be quantified(second biological substance) that is expressed on the detectionsubject.

5. Determination of Expression Amount of Substance to be Quantified(Second Biological Substance)

The amount of the substance to be quantified (second biologicalsubstance) that is expressed on the detection subject is determined bymeasuring the fluorescence intensity of the thus specified stained imageof the second immunostaining. In this case, “measuring the fluorescenceintensity” refers to measuring the number of bright spots or thefluorescence brightness for the thus specified bright spots orbrightness distribution.

The number of bright spots and the fluorescence brightness can bemeasured by a commonly used method. For example, the number of brightspots or the fluorescence brightness is measured by loading the stainedimage of the second immunostaining to a computer and performing imageprocessing by an arithmetic means using an analysis software for thespecified bright spots or brightness distribution. Examples of theanalysis software include “ImageJ”, which is a commercially availableimage analysis software, and “G-Count”, which is an automatic totalbright spot measuring software manufactured by G-Angstrom K.K.

By comparing the thus measured number of bright spots or fluorescencebrightness between samples or between a sample and a reference sample,the amount of the substance to be quantified that is expressed on thedetection subject in a sampled tissue section can be determined.

EXAMPLES

The present invention will now be described more concretely by way ofexamples thereof; however, the scope of the present invention is notrestricted thereto.

Example 1 (1) Bright-Field Observable Immunostaining on First BiologicalSubstance (First Immunostaining)

After immersing a liver tissue slide (T032a, manufactured by Biomax,Inc.) in xylene to remove paraffin, the liver tissue slide wasautoclaved in a citrate buffer (pH 6.0) for 15 minutes. Then, the slidewas washed with PBS, and 10% rabbit serum (manufactured by NichireiCorporation) was added thereto. The resulting slide was left to stand atroom temperature for 1 hour.

After washing this slide with PBS, an anti-CD31 antibody (mouseantibody, manufactured by Abcam plc.) was added thereto, and the slidewas left to stand at room temperature for 30 minutes. The slide wasagain washed with PBS and, after adding thereto a dextran polymerperoxidase-labeled anti-mouse antibody (manufactured by NichireiCorporation), the slide was left to stand at room temperature for 30minutes. Using a DAB substrate kit (manufactured by NichireiCorporation), the slide was immunostained with an enzyme anddiaminobenzidine (DAB) as a chromogenic substrate, followed by washingwith PBS.

The slide was sequentially immersed in ethanol and xylene and, afteradding thereto a mounting medium (Entellan New, manufactured by MerckKGaA) , a cover glass was placed on the slide to prepare an evaluationslide.

(2) Immunostaining on Second Biological Substance with FluorescentDye-Containing Nanoparticle (Second Immunostaining)

(a) Preparation of Antibody-Bound Fluorescent Melamine Resin Particle(Average Particle Size: 150 nm)

After adding and dissolving 14.4 mg SulfoRhodamine 101 (manufactured bySigma-Aldrich) in 22 mL of water, 2 mL of 5% aqueous solution of EMULGEN430 (manufactured by Kao Corporation), was added. The resulting solutionwas heated to 70° C. with stirring on a hot stirrer, and 0.65 g of amelamine resin material, NIKALAC MX-035 (manufactured by Nippon CarbideIndustries Co., Ltd.), was added thereto. Then, 680 μL of 10% aqueoussolution of dodecylbenzenesulfonic acid (manufactured by Kanto ChemicalCo., Inc.) was further added, and the resulting solution was heated withstirring at 70° C. for 50 minutes. Thereafter, this solution was furtherheated with stirring at 90° C. for 20 minutes. The resultingparticle-containing solution was washed with pure water so as to removeimpurities such as excess resin material and dye. This solution wascentrifuged at 20,000 G for 15 minutes using a centrifugal machine(micro-refrigerated centrifuge 3740, manufactured by Kubota Corporation)and, after removing the resulting supernatant, the precipitates werere-dispersed in ultrapure water by ultrasonication. The centrifugation,the removal of supernatant and the re-dispersion in ultrapure water wererepeated five times.

Then, 0.1 mg of the thus obtained particles were dispersed in 1.5 mL ofEtOH, and 2 μL of aminopropyltrimethoxysilane, LS-3150 (manufactured byShin-Etsu Chemical Co., Ltd.), was added thereto. The resulting mixturewas allowed to react for 8 hours to perform a surface aminationtreatment.

The thus obtained dye-containing nanoparticles were adjusted with PBS(phosphate-buffered physiological saline) containing 2 mM of EDTA(ethylenediamine tetraacetic acid) to a concentration of 3 nM. Thissolution was mixed with SM(PEG)12(succinimidyl-[(N-maleomidopropionamid)-dodecaethylene glycol]ester,manufactured by Thermo Fisher Scientific K.K.) to a final concentrationof 10 mM and allowed to react for 1 hour. The resulting mixture wascentrifuged at 10,000 G for 20 minutes and the resulting supernatant wasremoved. Then, PBS containing 2 mM of EDTA was added to disperse theprecipitates, and the resulting dispersion was centrifuged again. Theprecipitates were washed three times by the same procedure to obtainfluorescent dye-containing particles having a maleimide group at aterminal.

Meanwhile, streptavidin (manufactured by Wako Pure Chemical Industries,Ltd.) was subjected to a thiol group addition treatment withN-succinimidyl-S-acetylthioacetate (SATA), and the resultant wasfiltered through a gel-filtration column to obtain a solution ofstreptavidin capable of binding to dye-containing nanoparticles.

The above-described fluorescent nanoparticles and streptavidin weremixed in PBS containing 2 mM of EDTA and allowed to react for 1 hour.Then, the reaction was terminated with an addition of 10 mMmercaptoethanol. After concentrating the resulting solution using acentrifugation filter, unreacted streptavidin and the like were removedusing a purification gel-filtration column, thereby obtainingstreptavidin-bound SulfoRhodamine 101 dye-containing melaminenanoparticles.

(b) Preparation of Pathological Stain Solution

The thus obtained streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles (average particle size: 150 nm) were added to PBSat a concentration of 0.06 nM to prepare a pathological stain solution.

(c) Immunostaining

A liver tissue slide of a tissue section adjacent to the tissue sectionof the above-described liver tissue slide of (1) was immersed in xyleneto remove paraffin, and this liver tissue slide was subsequentlyautoclaved in a citrate buffer (pH 6.0) for 15 minutes. Then, the slidewas washed with PBS, 10% goat serum (manufactured by NichireiCorporation) was added thereto, and the resulting slide was left tostand at room temperature for 1 hour. After washing this slide with PBS,an anti-VEGFR-2 antibody (rabbit antibody, manufactured by Abcam plc.)was added thereto, and the slide was left to stand at room temperaturefor 30 minutes. The slide was again washed with PBS and, after addingthereto a biotin-labeled anti-rabbit antibody (manufactured by NichireiCorporation), the slide was left to stand at room temperature for 30minutes. To this slide, the above-prepared pathological stain solutionwas added after dilution and allowed to react at room temperature for 2hours, and the slide was subsequently washed with PBS.

The slide was sequentially immersed in ethanol and xylene and, afteradding thereto a mounting medium (Entellan New, manufactured by MerckKGaA) , a cover glass was placed on the slide to prepare an evaluationslide.

(3) Evaluation of Stained Images

For the thus prepared evaluation slides, a stained image of the firstimmunostaining was obtained under a light microscope (manufactured byCarl Zeiss AG) and a fluorescently stained image of the secondimmunostaining was obtained under a fluorescence microscope(manufactured by Carl Zeiss AG), respectively. In the acquisition of thefluorescently stained image, the excitation wavelength was 575 to 600 nmand the fluorescence wavelength was 612 to 682 nm.

The thus obtained stained image of the first immunostaining andfluorescently stained image of the second immunostaining were evaluated.The results thereof are shown in Table 1-1.

Example 2

An evaluation slide was prepared in the same manner as in Example 1except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 3

Evaluation slides were prepared in the same manner as in Example 1except that the first immunostaining was performed using an anti-CD34antibody (mouse antibody, manufactured by Nichirei Corporation), and thethus obtained stained image of the first immunostaining andfluorescently stained image of the second immunostaining were evaluated.The results thereof are shown in Table 1-1.

Example 4

An evaluation slide was prepared in the same manner as in Example 3except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 5

Evaluation slides were prepared in the same manner as in Example 1except that the second immunostaining was performed using ananti-VEGFR-1 antibody (rabbit antibody, manufactured by Abcam plc.), andthe thus obtained stained image of the first immunostaining andfluorescently stained image of the second immunostaining were evaluated.The results thereof are shown in Table 1-1.

Example 6

An evaluation slide was prepared in the same manner as in Example 5except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 7

Evaluation slides were prepared in the same manner as in Example 3except that the second immunostaining was performed using ananti-VEGFR-1 antibody (rabbit antibody, manufactured by Abcam plc.), andthe thus obtained stained image of the first immunostaining andfluorescently stained image of the second immunostaining were evaluated.The results thereof are shown in Table 1-1.

Example 8

An evaluation slide was prepared in the same manner as in Example 7except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 9

Evaluation slides were prepared in the same manner as in Example 1except that the first immunostaining was performed using ananti-podoplanin antibody (mouse antibody, manufactured by Medical &Biological Laboratories Co., Ltd.) and the second immunostaining wasperformed using an anti-VEGFR-1 antibody (rabbit antibody, manufacturedby Abcam plc.), and the thus obtained stained image of the firstimmunostaining and fluorescently stained image of the secondimmunostaining were evaluated. The results thereof are shown in Table1-1.

Example 10

An evaluation slide was prepared in the same manner as in Example 9except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 11

Evaluation slides were prepared in the same manner as in Example 1except that the first immunostaining was performed using ananti-cytokeratin AE1/AE3 antibody (mouse antibody, manufactured by DakoCo., Ltd.) and the second immunostaining was performed using ananti-Ki67 antibody (rabbit antibody, manufactured by NichireiCorporation), and the thus obtained stained image of the firstimmunostaining and fluorescently stained image of the secondimmunostaining were evaluated. The results thereof are shown in Table1-1.

Example 12

An evaluation slide was prepared in the same manner as in Example 11except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 13

Evaluation slides were prepared in the same manner as in Example 11except that the second immunostaining was performed using an anti-ERantibody (rabbit antibody, manufactured by Nichirei Corporation), andthe thus obtained stained image of the first immunostaining andfluorescently stained image of the second immunostaining were evaluated.The results thereof are shown in Table 1-1.

Example 14

An evaluation slide was prepared in the same manner as in Example 13except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 15

Evaluation slides were prepared in the same manner as in Example 11except that the second immunostaining was performed using an anti-PgRantibody (rabbit antibody, manufactured by Ventana Medical Systems,Inc.), and the thus obtained stained image of the first immunostainingand fluorescently stained image of the second immunostaining wereevaluated. The results thereof are shown in Table 1-1.

Example 16

An evaluation slide was prepared in the same manner as in Example 15except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 17

Evaluation slides were prepared in the same manner as in Example 1except that the first immunostaining was performed using an anti-CK7antibody (mouse antibody, manufactured by Acris Antibodies GmbH) , andthe thus obtained stained image of the first immunostaining andfluorescently stained image of the second immunostaining were evaluated.The results thereof are shown in Table 1-1.

Example 18

An evaluation slide was prepared in the same manner as in Example 17except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 19

Evaluation slides were prepared in the same manner as in Example 17except that the second immunostaining was performed using ananti-VEGFR-1 antibody (rabbit antibody, manufactured by Abcam plc.), andthe thus obtained stained image of the first immunostaining andfluorescently stained image of the second immunostaining were evaluated.The results thereof are shown in Table 1-1.

Example 20

An evaluation slide was prepared in the same manner as in Example 19except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Example 21

Evaluation slides were prepared in the same manner as in Example 17except that the second immunostaining was performed using ananti-VEGFR-3 antibody (rabbit antibody, manufactured by Abcam plc.), andthe thus obtained stained image of the first immunostaining andfluorescently stained image of the second immunostaining were evaluated.The results thereof are shown in Table 1-1.

Example 22

An evaluation slide was prepared in the same manner as in Example 21except that the second immunostaining was performed on the same livertissue slide as the first immunostaining, and the thus obtained stainedimage of the first immunostaining and fluorescently stained image of thesecond immunostaining were evaluated. The results thereof are shown inTable 1-1.

Comparative Example 1

An evaluation slide was prepared by performing only the secondimmunostaining in Example 1 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 2

An evaluation slide was prepared in the same manner as in Example 2except that the second immunostaining was performed usingStreptavidin-Alexa Fluor (registered trademark) 594 conjugate(manufactured by Invitrogen) in place of the streptavidin-boundSulfoRhodamine 101 dye-containing melamine nanoparticles. The secondimmunostaining using Streptavidin-Alexa Fluor 594 conjugate(manufactured by Invitrogen) was performed in accordance with theproduct manual provided by Invitrogen. The thus obtained evaluationslide was evaluated. The results thereof are shown in Table 1-2.

Comparative Example 3

An evaluation slide was prepared by performing only the secondimmunostaining in Example 3 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 4

An evaluation slide was prepared in the same manner as in Example 4except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 5

An evaluation slide was prepared by performing only the secondimmunostaining in Example 5 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 6

An evaluation slide was prepared in the same manner as in Example 6except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 7

An evaluation slide was prepared by performing only the secondimmunostaining in Example 7 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 8

An evaluation slide was prepared in the same manner as in Example 8except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 9

An evaluation slide was prepared by performing only the secondimmunostaining in Example 9 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 10

An evaluation slide was prepared in the same manner as in Example 10except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 11

An evaluation slide was prepared by performing only the secondimmunostaining in Example 11 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 12

An evaluation slide was prepared in the same manner as in Example 12except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 13

An evaluation slide was prepared by performing only the secondimmunostaining in Example 13 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 14

An evaluation slide was prepared in the same manner as in Example 14except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 15

An evaluation slide was prepared by performing only the secondimmunostaining in Example 15 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 16

An evaluation slide was prepared in the same manner as in Example 16except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 17

An evaluation slide was prepared by performing only the secondimmunostaining in Example 17 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 18

An evaluation slide was prepared in the same manner as in Example 18except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-boundSulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 19

An evaluation slide was prepared by performing only the secondimmunostaining in Example 19 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 20

An evaluation slide was prepared in the same manner as in Example 20except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-bound SulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

Comparative Example 21

An evaluation slide was prepared by performing only the secondimmunostaining in Example 21 without the first immunostaining. Theresults of evaluating the thus obtained stained image are shown in Table1-2.

Comparative Example 22

An evaluation slide was prepared in the same manner as in Example 22except that the second immunostaining was performed usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) inplace of the streptavidin-boundSulfoRhodamine 101 dye-containingmelamine nanoparticles. The second immunostaining usingStreptavidin-Alexa Fluor 594 conjugate (manufactured by Invitrogen) wasperformed by the method described in Comparative Example 2. The thusobtained evaluation slide was evaluated. The results thereof are shownin Table 1-2.

[Evaluation Results of Evaluation Slides]

As shown in Table 1, even when the evaluation slides of differentadjacent tissue sections were used, the expression positions of thesecond biological substance on the specific tissue or cells (thepositions of the bright spots and the brightness distribution on therespective fluorescently stained images of the second immunostaining)were specified by comparing the stained image of the firstimmunostaining and the stained image of the second tissue section(Examples). Moreover, when the evaluation slides of the same tissuesection were used, the expression positions of the second biologicalsubstance were easily and accurately specified without having to changeslides (Examples). On the other hand, when the first immunostaining wasnot performed, it was difficult to specify the expression positions ofthe second biological substance on the specific tissue or cells(Comparative Examples).

Furthermore, with regard to the expression amount of the secondbiological substance (calculation of the number of bright spots anddetermination of the brightness distribution on the respectivefluorescently stained images of the second immunostaining), when afluorescent dye was used for the second immunostaining, the fluorescenceintensity was low and it was thus difficult to determine thefluorescence brightness distribution (Comparative Examples); however,when fluorescent dye-containing nanoparticles were used, a sufficientfluorescence intensity was obtained and the fluorescence brightnessdistribution was thus determined with no problem (Examples).

TABLE 1-1 Specification of Determination of expression posi- brightnessdistri- Tissue tions of second bution of second section biologicalsubstance biological substance First immunostaining Secondimmunostaining slide (Note 1) (Note 2) Example 1 CD31-DABVEGFR2-fluorescent dye-containing adjacent ◯ ⊚ nanoparticle Example 2CD31-DAB VEGFR2-fluorescent dye-containing same ⊚ ⊚ nanoparticle Example3 CD34-DAB VEGFR2-fluorescent dye-containing adjacent ◯ ⊚ nanoparticleExample 4 CD34-DAB VEGFR2-fluorescent dye-containing same ⊚ ⊚nanoparticle Example 5 CD31-DAB VEGFR1-fluorescent dye-containingadjacent ◯ ⊚ nanoparticle Example 6 CD31-DAB VEGFR1-fluorescentdye-containing same ⊚ ⊚ nanoparticle Example 7 CD34-DABVEGFR1-fluorescent dye-containing adjacent ◯ ⊚ nanoparticle Example 8CD34-DAB VEGFR1-fluorescent dye-containing same ⊚ ⊚ nanoparticle Example9 Podoplanin-DAB VEGFR3-fluorescent dye-containing adjacent ◯ ⊚nanoparticle Example 10 Podoplanin-DAB VEGFR3-fluorescent dye-containingsame ⊚ ⊚ nanoparticle Example 11 Cytokeratin AE1/AE3-DABKi67-fluorescent dye-containing adjacent ◯ ⊚ nanoparticle Example 12Cytokeratin AE1/AE3-DAB Ki67-fluorescent dye-containing same ⊚ ⊚nanoparticle Example 13 Cytokeratin AE1/AE3-DAB ER-fluorescentdye-containing adjacent ◯ ⊚ nanoparticle Example 14 CytokeratinAE1/AE3-DAB ER-fluorescent dye-containing same ⊚ ⊚ nanoparticle Example15 Cytokeratin AE1/AE3-DAB PgR-fluorescent dye-containing adjacent ◯ ⊚nanoparticle Example 16 Cytokeratin AE1/AE3-DAB PgR-fluorescentdye-containing same ⊚ ⊚ nanoparticle Example 17 CK7-DABVEGFR2-fluorescent dye-containing adjacent ◯ ⊚ nanoparticle Example 18CK7-DAB VEGFR2-fluorescent dye-containing same ⊚ ⊚ nanoparticle Example19 CK7-DAB VEGFR1-fluorescent dye-containing adjacent ◯ ⊚ nanoparticleExample 20 CK7-DAB VEGFR1-fluorescent dye-containing same ⊚ ⊚nanoparticle Example 21 CK7-DAB VEGFR3-fluorescent dye-containingadjacent ◯ ⊚ nanoparticle Example 22 CK7-DAB VEGFR3-fluorescentdye-containing same ⊚ ⊚ nanoparticle

TABLE 1-2 Specification of Determination of expression posi- brightnessdistri- Tissue tions of second bution of second section biologicalsubstance biological substance First immunostaining Secondimmunostaining slide (Note 1) (Note 2) Comparative noneVEGFR2-fluorescent dye-containing — X ◯ Example 1 nanoparticleComparative CD31-DAB VEGFR2-fluorescent dye same ⊚ X Example 2Comparative none VEGFR2-fluorescent dye-containing — X ◯ Example 3nanoparticle Comparative CD34-DAB VEGFR2-fluorescent dye same ⊚ XExample 4 Comparative none VEGFR1-flucrescent dye-containing — X ◯Example 5 nanoparticle Comparative CD31-DAB VEGFR1-fluorescent dye same⊚ X Example 6 Comparative none VEGFR1-fluorescent dye-containing — X ◯Example 7 nanoparticle Comparative CD34-DAB VEGFR1-fluorescent dye same⊚ X Example 8 Comparative none VEGFR3-fluorescent dye-containing — X ◯Example 9 nanoparticle Comparative podoplanin-DAB VEGFR3-fluorescent dyesame ⊚ X Example 10 Comparative none Ki67-fluorescent dye-containing — X◯ Example 11 nanoparticle Comparative Cytokeratin AE1/AE3-DABKi67-fluorescent dye same ⊚ X Example 12 Comparative none ER-fluorescentdye-containing — X ◯ Example 13 nanoparticle Comparative CytokeratinAE1/AE3-DAB ER-fluorescent dye same ⊚ X Example 14 Comparative nonePgR-fluorescent dye-containing — X ◯ Example 15 nanoparticle ComparativeCytokeratin AE1/AE3-DAB PgR-fluorescent dye same ⊚ X Example 16Comparative none VEGFR2-fluorescent dye-containing — X ◯ Example 17nanoparticle Comparative CK7-DAB VEGFR2-fluorescent dye same ⊚ X Example18 Comparative none VEGFR1-fluorescent dye-containing — X ◯ Example 19nanoparticle Comparative CK7-DAB VEGFR1-fluorescent dye same ⊚ X Example20 Comparative none VEGFR3-fluorescent dye-containing — X ◯ Example 21nanoparticle Comparative CK7-DAB VEGFR3-fluorescent dye same ⊚ X Example22

-   (Note 1) Specification of the expression positions of the second    biological substance on the specific tissue or cells based on a    comparison between the stained image of the first immunostaining and    that of the second tissue section (the positions of bright spots and    the brightness distribution on the respective fluorescently stained    images of the second immunostaining):

⊚: accurately and easily specifiable

∘: specifiable

×: not specifiable

-   (Note 2) Determination of the expression amount of the second    biological substance (calculation of the number of bright spots and    determination of the brightness distribution on the respective    fluorescently stained images of the second immunostaining):

⊚: The brightness distribution could be determined and the positions ofbright spots were specifiable.

∘: The brightness distribution could be determined, but the positions ofbright spots could not be specified.

×: The brightness distribution could not be determined.

DESCRIPTION OF SYMBOLS

1: Vascular endothelial cell

2: VEGFR2

1. A method of quantifying a biological substance in a tissue section,said method comprising: (1) performing bright-field observableimmunostaining that specifically stains a first biological substance insaid tissue section (first immunostaining); (2) performingimmunostaining with a fluorescent substance-containing nanoparticle thatspecifically stains a second biological substance in said tissue section(second immunostaining); (3) specifying the expression position(s) ofsaid second biological substance in said tissue section by comparing theposition of a stained image of said first immunostaining and position ofa stained image of said second immunostaining; and (4) determining theexpression amount of said second biological substance by measuring thefluorescence intensity of said stained image of said secondimmunostaining.
 2. The method according to claim 1, wherein saidfluorescence intensity of said stained image of said secondimmunostaining is measured at a position(s) where said stained image ofsaid first immunostaining and that of said second immunostaining overlapwith each other.
 3. The method according to claim 1, wherein said firstimmunostaining and said second immunostaining are performed on the sametissue section.